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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver.
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Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver.

机译：肝炎病毒感染的肝脏中共价闭合的环状DNA拷贝数的单细胞分析。

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摘要

Hepatitis B virus (hepadnavirus) infections are maintained by the presence of a small and regulated number of episomal viral genomes [covalently closed circular DNA (cccDNA)] in the nuclei of infected cells. Although a number of studies have measured the mean copy number of cccDNA molecules in hepadnaviral-infected cells, the distribution of individual copy numbers have not been reported. Using a PCR-based assay, we examined the number of cccDNA molecules of the duck hepatitis B virus in single nuclei isolated from the liver of a chronically infected duck over the course of 131 days of infection. Nuclei were isolated from frozen serial biopsies and individually deposited into PCR microplates by flow sorting. Each nucleus was assayed by nested PCR for cccDNA and for cellular IFN-alpha genes as an internal control. We found that 90% of the nuclei assayed contained between 1 and 17 cccDNA molecules, with the remaining 10% containing more (90% confidence), and that differences in the mean number of copies and distribution of copy numbers occurred within the same animal at different times postinfection. Overall, the data suggest (i) that the number of cccDNA molecules per cell may fluctuate over time, and (ii) that, according to these fluctuations, a substantial fraction of cells may contain only one or a few copies. We infer from the results that infected hepatocytes express virus at different levels and that during cell division it is possible to segregate cells containing no cccDNA.
机译：乙型肝炎病毒（肝炎病毒）感染是通过在感染细胞核中存在少量且受调控的游离病毒基因组[共价闭合环状DNA（cccDNA）]来维持的。尽管许多研究测量了肝炎病毒感染细胞中cccDNA分子的平均拷贝数，但尚未报告单个拷贝数的分布。使用基于PCR的分析，我们在感染131天的过程中检查了从慢性感染鸭肝中分离出的单个核中鸭乙型肝炎病毒cccDNA分子的数量。从冷冻连续活检组织中分离细胞核，并通过流分选分别沉积到PCR微孔板中。通过巢式PCR检测每个核的cccDNA和细胞IFN-α基因作为内部对照。我们发现，所分析的90％的核包含1至17个cccDNA分子，其余10％的核包含更多（90％的置信度），并且同一动物中平均拷贝数和拷贝数分布存在差异感染后的不同时间。总的来说，数据表明（i）每个细胞的cccDNA分子数量可能会随时间波动，并且（ii）根据这些波动，很大一部分细胞可能仅包含一个或几个拷贝。我们从结果中推断出感染的肝细胞以不同水平表达病毒，并且在细胞分裂过程中可以分离不含cccDNA的细胞。

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